It has been known for almost a century that embryo transfer is one tool for assisting in the genetic improvement of livestock. Embryo transfer occurs after embryo division, and the ultimate result is the production of twin animals having similar and enhanced genetic qualities. For example, naturally occurring twin bovine offspring are generally regarded as undesirable and are infrequently genetically identical. In addition, if the twin offspring consists of a male and a female, the male influences the female to such a degree as to produce a sterile female called a freemartin. However, twin bovine offspring produced by embryo splitting and embryo transfer are distinct from naturally occurring twins in that they both have the same sex and do not suffer the disadvantages of sterility. Among the advantages of embryo splitting as the preferred form of bovine cloning are more pregnancies per donor recovery and the absence of long-term culture (5-7 days) of the bovine embryo prior to transfer to a recipient. This can also provide a quicker return on investment by reducing the cost of embryo transfer, and allowing half of the embryo to be transferred and half to be used in sexing (splitting only female embryos) or freezing.
Since the 1970's embryo splitting and embryo transfer have become an established practice in livestock production.
Techniques for embryo splitting and embryo transfer originated with work on laboratory animals. Because the size of the embryos undergoing division are often between 70 and 150 microns in diameter, the precision manipulations on the embryos are referred to as micromanipulations.
Experimentation over the last two decades has resulted in the utilization of a number of different techniques for dividing embryos. Common to all techniques are at least four essentials:
1) a medium for maintaining the embryos during micromanipulation; PA1 2) a means of holding the embryo; PA1 3) a means for holding the micro-instrument(s) used in embryo division; and PA1 4) a controlled means of bringing the embryo and the micro-instrument(s) together. PA1 1) blastomere separation wherein the early cleavage stage blastomeres are not firmly bound one to another; PA1 2) division of pre-compaction morulae wherein the zona pellucida is opened by a fine glass needle or a horizontal blade, and the morula is expelled or withdrawn by an aspiration pipette. The withdrawn morula are then inserted into a surrogate zona pellucida; and PA1 3) division of post-compaction morulae and blastocysts wherein either the blade or needle method is employed to divide the embryo while it is in the zona pellucida or after it has been withdrawn.
A common holding medium is a phosphate-buffered saline (PBS) medium. Included in this medium may be one to ten percent of heat-treated fetal calf serum (FCS). In addition, antibiotics such as penicillin, streptomycin, or amphotericin are added as well as physiological concentrations of glucose or sodium pyruvate. The embryos and the maintaining medium are contained in a sterile, plastic petri dish, and the micromanipulations on the embryo(s) are performed while they are maintained in the petri dish or on a depression slide.
Several key terms which occur in the micromanipulation of embryos should be defined for the purpose of clarity and understanding. The term "embryo" is a loose term and connotes any of the various early developmental stages of mammals. Depending on the circumstance, the "embryo" could denote a zygote, a morula, or a blastocyst. The zona pellucida is the acellular glycoprotein material that forms around the oocyte in the ovary and continues to surround the embryo until the blastocyst hatches from it. Embryos that reach the 16-cell stage begin to resemble mulberries and are called morulae. A blastomere--or cleavage cell--is one of the cells into which the egg divides after fertilization, and a blastocyst consists of an inner cell mass and a thin outer trophoblast layer, the blastocyst being a modified early stage of embryo development. Furthermore, there are generally three categories of embryo division:
Several different methods for dividing embryos are practiced in medical laboratories, research facilities, and veterinary practices. The micromanipulators are the pieces of equipment or devices which allow the precision, superfine manipulation of the micro-instruments used for dividing embryos, i.e., the microneedles and/or microblades used for embryo dividing. Pneumatic, slide-rail, sliding, and electrically-controlled wormgear are among the various kinds of micromanipulators currently in use.
One method to divide an embryo is to hold it by suction against a fire-polished holding pipette. The holding pipette is controlled by a micromanipulator, and the suction is such that the zona pellucida bulges slightly into the holding pipette. On a second micromanipulator, immediately adjacent the micromanipulator carrying the holding pipette, is the instrument for dividing the embryo. The instrument can be a superfine needle for opening the zona pellucida and dividing the embryo or a horizontal blade glued to the outside of a glass pipette or a vertical blade glued inside the lumen of capillary tubing. The micromanipulator carrying the instrument for splitting the embryo must be of extremely high quality since the movements of the instrument are on the micron range. Then, with the embryo secured by the holding pipette and located near the bottom of the petri dish, the embryo is divided by the lateral movement of the vertical blade or the downward movement of the horizontal blade. Each half of the bisected embryo, now referred to as a "half embryo" or "demi-embryo", is then immediately transferred to a fresh holding medium with, for example, a sterile Pasteur pipette.
The embryos can also be immobilized on a slide surface or on the bottom of a petri dish instead of being held with suction by a holding pipette. In addition, a stereomicroscope or a compound microscope with a magnification potential of 100.times. is also used to assist the researcher in splitting the embryo. Since embryos can be 70 to 150 microns in diameter, the unaided eye would be completely unable to guide the instruments carried by the micromanipulator for embryo splitting.
Another method of dividing embryos is by using a pivoting microscalpel blade with a cutting edge 10 to 30 microns thick carried on, and secured to, a micromanipulator. With this method the zona pellucida is cut open by a pair of glass microneedles, then a micropipette is inserted through the slit, and the embryo is ejected by dispersal of a small volume of a medium into the zona pellucida. A microscalpel then bisects the embryo (in its blastocyst stage) along its sagittal plane. Each half embryo (demi-embryo) is then placed in an empty zona pellucida (which has been opened and emptied of its embryo prior to embryo splitting) by a micropipette using the aforementioned technique.
Among the factors affecting the ultimate viability of the embryo after division is the quality of the embryos chosen for splitting and the skill in operating the micromanipulators and the micro-instruments to achieve properly bisected embryos. Embryos should have a large number of viable cells and be in the late morula or early blastocyst stage. In achieving proper embryo bisecting, acute skill in using the aforementioned instruments is critical and vital for the procedure to be executed successfully.